Enhanced neuronal activity by suffruticosol A extracted from Paeonia lactiflora via partly BDNF signaling in scopolamine-induced memory-impaired mice

Neurodegenerative diseases are explained by progressive defects of cognitive function and memory. These defects of cognition and memory dysfunction can be induced by the loss of brain-derived neurotrophic factors (BDNF) signaling. Paeonia lactiflora is a traditionally used medicinal herb in Asian countries and some beneficial effects have been reported, including anti-oxidative, anti-inflammatory, anti-cancer activity, and potential neuroprotective effects recently. In this study, we found that suffruticosol A is a major compound in seeds of Paeonia lactiflora. When treated in a SH-SY5 cell line for measuring cell viability and cell survival, suffruticosol A increased cell viability (at 20 µM) and recovered scopolamine-induced neurodegenerative characteristics in the cells. To further confirm its neural amelioration effects in the animals, suffruticosol A (4 or 15 ng, twice a week) was administered into the third ventricle beside the brain of C57BL/6 mice for one month then the scopolamine was intraperitoneally injected into these mice to induce impairments of cognition and memory before conducting behavioral experiments. Central administration of suffruticosol A into the brain restored the memory and cognition behaviors in mice that received the scopolamine. Consistently, the central treatments of suffruticosol A showed rescued cholinergic deficits and BDNF signaling in the hippocampus of mice. Finally, we measured the long-term potentiation (LTP) in the hippocampal CA3–CA1 synapse to figure out the restoration of the synaptic mechanism of learning and memory. Bath application of suffruticosol A (40 µM) improved LTP impairment induced by scopolamine in hippocampal slices. In conclusion, the central administration of suffruticosol A ameliorated neuronal effects partly through elevated BDNF signaling.


Materials and methods
Isolation of resveratrol oligomer from Paeonia lactiflora seed. Plant material. The seeds of P. lactiflora were harvested from the herb garden of the Medicinal Plant Experiment Station, located in Uisong (Korea), and were identified by one of the authors (Dr. C. W. Choi, GBSA, Suwon). The collection of plant seeds complied with relevant institutional, national, and international guidelines and legislation. A voucher specimen (KR1072) was estimated in the Korea Research Institute of Chemical Technology (KRICT) and deposited at its herbarium.
Extraction and isolation. Dried seeds of P. lactiflora (2 kg) were extracted with 10 L of mixed (70% EtOH) and then evaporated to dryness, resulting in 201 g of dark syrupy extract. This extract was suspended in H 2 O (5 L) and separated consequently with an equal volume of ethyl acetate (EtOAc), n-butanol (n-BuOH), and n-hexane, yielding an EtOAc soluble fraction (128.6 g), a n-BuOH soluble fraction (13.6 g), a n-hexane soluble fraction (21.9 g) and a residual aqueous fraction (34.9 g). The EtOAc soluble fraction (128.6 g) was placed in silica gel (1.5 kg) column (Ø = 8.0 × 60 cm) chromatography, eluted with MeOH in CH 2 Cl 2 according to a step-gradient manner (1% to 50%) to give six fractions (F1: 12.2 g, F2: 3.2 g, F3: 13.0 g, F4: 73.5 g, F5: 16.5 g and F6: 6.7 g). Finally, we achieved the purification of suffruticosol A (31 g) from repeated RP-18 chromatography of F4 with step-gradient elution of MeOH in H 2 O (Fig. 1A).  Cell viability experiment. Following the treatment, cell viability, and cell survival effects against scopolamine were assessed by MTT Assay Kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. Briefly, the cell media and treatment were gently discarded using a micropipette to minimize any potential color interference of suffruticosol A. Next, 50 μL of serum-free medium and 50 μL of MTT reagent were added to each well. After applying the MTT solution, the cells were incubated for 3 h, followed by the addition of 150 μL of MTT solvent to each well. The plate was wrapped in foil to prevent light exposure and shaken using an orbital shaker. The absorbance was measured at 590 nm using a Cannulation procedure and treatments. The mice underwent cannula implantation into the hypothalamic third ventricle (3 V) using a modified version of the procedure described previously 33 . A 26-gauge guide cannula (Plastics One, Roanoke, VA, USA) was implanted using an ultraprecise small animal stereotactic apparatus (Kopf Instruments, Tujunga, CA, USA). The coordinates for cannula implantation were 1.5 mm posterior to the bregma and 5.0 mm below the bregma. All mice underwent cannulation regardless of the treatment. Following the surgery, the animals had a one-week recovery period. The animals were randomly divided into the following groups (n = 5/group): Control (  34 . A white chamber measuring 40 cm in length, 40 cm in width, and 50 cm in height was used for the open field test. The mouse was placed in the center of the chamber and allowed to acclimate for 20 min. After 24 h of acclimation, all mice were placed in the same chamber for tracking and the total distance moved (in meters) was measured. Y-maze test: the Y-maze test was performed as previously described 35 .
The Y-maze consisted of three identical arms measuring 40 cm in length, 4 cm in width, and 15 cm in height, arranged at 120° angles. Visual cues were placed at the end of each arm. The mouse was gently placed at the end of one arm with its head facing away from the center. The mouse was then allowed to freely explore all three arms for 10 min. During the exploration, the number of entries into each arm was recorded. Spontaneous alternation was measured as sequential entries into all three arms (e.g., ABC, BCA, CAB, but not ABA), excluding repeated entries into the same arm. The percentage of spontaneous alternation was calculated using the following formula: Percentage = [(Number of spontaneous alternations)/(Total arm entries − 2)] × 100. Passive Avoidance Test: the passive avoidance test was performed as described previously 36 . The test was conducted using an Avoidance System (B.S. Technolab INC., Seoul, Korea). The apparatus consisted of two chambers, a light chamber, and a dark chamber, separated by a gate in the middle. For acclimation, each mouse was placed in the light chamber with the gate open and allowed to freely explore both chambers for 10 min. After 24 h, a training session was conducted. The mouse was gently placed in the light chamber with the door closed, and after 60 s, the gate was opened. The step-through latency, or the time taken for the mouse to enter the dark chamber, was recorded. Once the mouse entered the dark chamber, the gate was closed and a mild electrical shock (0.3 mA, 3 s) was applied to the mouse's foot. On the following day (probe trial), the mouse was placed in the light chamber with the door closed, and after 60 s, the gates were opened. The step-through latency was recorded for up to 300 s.

Electrophysiology.
Adult mice (C57/BL6J, age 5-6 weeks) were anesthetized using isoflurane. The brain was rapidly removed and immersed in an ice-cold oxygenated sucrose-based dissection buffer containing the following concentrations (in mM): 1.23 NaH 2 PO 4 , 5 KCl, 0.5 CaCl 2 , 26 NaHCO 3 , 10 MgSO 4, and 212.5 sucrose saturated with 5% CO 2 -95% O 2 , at pH 7.4. The brain was then mounted on the stage of a vibrating microtome (DSK Linear Slicer NLS-MT, Kyoto, Japan), and transverse slices of 400 µm thickness were sectioned. The slices were then transferred to an incubation chamber at room temperature and allowed to recover for one hour before recording, in standard oxygenated artificial cerebrospinal fluid (aCSF) composed of the following concentrations (in mM): 24 NaHCO 3 , 130 NaCl, 1.25 NaH 2 PO 4 , 3.5 KCl, 1.5 MgCl 2 , 1.5 CaCl 2 , and 10 glucose saturated with 95% O 2 -5% CO 2 , at pH 7.4. Slices of the hippocampus were mounted on the stage and superfused with oxygenated aCSF at 28 °C. A concentric bipolar electrode (CBBPE75, FHC, Bowdoin, ME, USA) stimulated the Schaffer collateral pathway, and we recorded field excitatory postsynaptic potential (fEPSP) from the stratum radiatum of CA1 using a glass pipette filled with aCSF (1-3 MΩ). The evoked fEPSP responses were intensified by a Multiclamp 700b (Axon Instruments, San Jose, CA, USA) and expressed by Digidata 1322A. The slope of the fEPSP response was calculated by using pCLAMP 10 software (Axon Instruments, San Jose, CA, USA). The stimulation intensity was attuned to obtain fEPSP slopes of 50-60% to the maximum. Bath temperature was maintained at 28 °C by a temperature controller (TC344B, Warner Instrument Corporation, Hamden, CT, USA) during recordings. The basal slope of the fEPSP was monitored by electrical stimulation at 0.1 Hz. For the longterm potentiation (LTP) induction, electrical stimulations were applied as theta-burst stimulation (TBS), con-  Statistical analysis. Statistical analysis and plot generation were conducted using R (R Core Team, 2021) with the "tidyverse", "rstatix", and "ggpubr" packages. The experimental values were visualized as mean ± standard error of the mean (S.E.M.) and differences among groups were evaluated with one-way ANOVA by Tukey's post hoc test. P-values less than 0.05 were considered significant.

Results
Identification of suffruticosol A from seeds of P. lactiflora. Specific features were observed in these compounds. The basis of the structure elucidation process was formed by analyzing the 1 H-and 13 C-NMR spectra of these compounds. The 1 H-NMR spectra revealed the presence of six sets of ortho-coupled aromatic hydrogens applied to three 4-hydroxyphenyl groups (A ring of a compound) and signals from three other 3,5-dihydroxy phenyl systems (B ring of a compound) distinctive for three resveratrol units. Instead of the signals for olefinic protons (cis or trans) of resveratrol units, the reduction of these olefinic bonds was recommended by the presence of six methine hydrogens strongly and their trimerization involving these carbons of the three resveratrol units. The analysis of 13  www.nature.com/scientificreports/ pounds are characterized as resveratrol trimers. In the spectrum results, it showed that the presence of the highly deshielded oxymethine (δ H ~ 6.00, δc ~ 90.0) of each of these three trimers revealed a dihydrofuran ring system. Interestingly, some distinct differences in 1 H-and 13 C-NMR signals of these three trimers were observed and this difference suggested that these structures of trimers are significantly different. Thus, based on those data and in comparison with literature 19 , the isolated compound was identified as suffruticosol A in Fig. 1B, C.

Suffruticosol A elevated viability and cell survival against scopolamine treatments in vitro.
Previous studies have reported therapeutic possibilities of resveratrol on neural activities in vitro 24 , but with insufficient results on its therapeutic effects 26 . Recent research has highlighted the potential of suffruticosol A, a resveratrol trimer, in exerting therapeutic effects against neuronal cell death 27 , which is associated with AD 28 , although evidence is limited. In this study, we utilized a human neuroblastoma cell line, SH-SY5Y, to evaluate the neural survival effects of suffruticosol A in the presence of scopolamine treatment. Prior to assessing the cell survival effects of this compound, we examined its impact on cell viability in SH-SY5Y cells. Treatments with suffruticosol A resulted in a dose-dependent increase in cell viability, with a significant increase observed at 20 μM ( Fig. 2A, F (

The central administration of suffruticosol A improved memory and cognitive behaviors.
Building upon the elevated neural activities observed with suffruticosol A in vitro, we aimed to investigate whether treatment with suffruticosol A via the third ventricle of the brain could enhance memory and cognition in experimental animals that received scopolamine to induce deficits in these functions. For accurate experimental procedures, we designed the schedules as depicted in Fig. 3A. Subsequently, stereotaxic surgery was performed to implant the cannula into the third ventricle of the brain. To determine the appropri- www.nature.com/scientificreports/ ate dose of suffruticosol A for central administration, we reviewed and analyzed several studies using central administration [37][38][39][40] . Based on these analyses, we modified the formula which we used in our previous studies for selecting the optimal dose for the brain 33,41 . Then, we applied the results of cell lines (from Fig. 2) to this formula for deciding the dose of central administration in animals 42 . Firstly, we converted the units from μM to ng/μl, rounded them to whole numbers, and then multiplied them by two, as demonstrated in Eq. (1).
Consequently, we administered a low dose of suffruticol A (Suff/L, 4 ng) or a high dose of suffruticosol A (Suff/H, 15 ng) through a cannula into the brains of the animals. Initially, we did central administration for 3 days for expecting the acute effect of this chemical. However, the results of Y-maze tests showed no significant changes by suffruticosol A treatment (data not shown). In this study, the central treatments of suffruticosol A were relatively low doses (4 ng or 15 ng), compared to those in other reference use (µg or mg) 37,39,40 . Thus, these low-dose treatments are suitable for inducing the chronic effects of suffruticosol A. To evaluate the long-term effect of suffruticosol A, we kept the frequency of central administration twice a week for one month. Following the chronic treatments of suffruticosol A (1 month), scopolamine (1.0 mg/kg) was intraperitoneally administered to the Veh, Suff/L, and Suff/H groups prior to the behavioral tests (30 min in advance). The scopolamine injection in mice leads to increased locomotion 43 . To confirm the proper action of scopolamine, we measured the locomotion of these experimental mice by open-field tests. The treatments of scopolamine induced significantly higher activities in the mice but treatments of suffruticosol A affected trends in increased locomotion without significance against the treatments of scopolamine (Fig. 3B, F (3,16) = 8.219, Con vs. Scop + Veh, p < 0.01). To measure shortterm memory, we tested a Y-maze test for analyzing spontaneous alternation. This alternation was inhibited by scopolamine treatments but protected in the high dose of suffruticosol A (Fig. 3C, F (3,12) = 9.776, Scop + Veh vs. Scop + Suff/H, p < 0.05). The total arm entries of all groups showed no differences (Fig. 3D, F (3,12) = 1.011, One-way Anova, p = 0.422) which refers lowered spontaneous alternation of Scop group is not derived from hyperactivity but from memory impairment. To examine whether the treatments of suffruticosol A had long-term effects on memory, we conducted passive avoidance tests on these mice. Consistently, scopolamine treatments impaired memory, while high doses of suffruticosol A induced the restoration of memory (Fig. 3E, F (3,19) = 4.838, Con vs. Scop + Veh, p < 0.05, Scop + Veh vs. Scop + Suff/H, p < 0.05). This suggests that the chronic treatments of suffruticosol A rescue neural activity against memory deficit induced by the scopolamine treatments.

Suffruticosol A restored the impairments of the cholinergic systems by the scopolamine treatments in the hippocampus.
Depletion of the cholinergic system leads to a reduced BDNF signaling, and these factors are associated with cognitive deficits 44 . To determine whether suffruticosol A treatments in the brain can restore cholinergic system activity, we assessed the activities of choline acetyltransferase (ChAT), acetylcholine contents (ACh), and acetylcholinesterase (AChE), which are crucial for neuronal functions. The treatments of scopolamine significantly decreased ACh levels and ChAT activities (Fig. 4A, B (Fig. 4A, B). In contrast, scopolamine treatments significantly increased AChE activity, but suffruticosol A treatments did not restore this activity (Fig. 4C, F (3,16) = 5.832).

Central administration of suffruticosol A elevates BDNF signaling.
To further elucidate the molecular mechanism underlying the restoration of neural activity, we analyzed the mRNA expression of Bdnf, TrkB, Akt, and Creb1. These signaling pathways are closely associated with cognition and memory function, and the treatments of scopolamine down-regulate BDNF signaling 10 . Mature BDNF (mBDNF) binds to the TrkB receptor, activating Akt and cAMP response element-binding protein (CREB) signaling, which enhances neuronal survival, growth, and synaptic plasticity regulation 45,46 . Therefore, Suffruticosol A treatments may restore the impaired BDNF signaling caused by scopolamine treatments. We extracted RNAs from the hippocampi to analyze the mRNA expression of the BDNF signaling cascades. Interestingly, the high-dose Suffruticosol A treatments significantly restored BDNF signaling that was impaired by scopolamine ( Fig. 5A-C). The mRNA expression of TrkB, Akt, and Creb1, which were down-regulated by scopolamine treatment, were rescued significantly in the Scop + Suff/H groups (5A: F (3,14) = 19.631, Scop + Veh vs. Scop + Suff/H, p < 0.01, 5B: F (3,14) = 22.845, Scop + Veh vs. Scop + Suff/H, p < 0.05, 5C: F (3,14) = 28.666, Scop + Veh vs. Scop + Suff/H, p < 0.05). The mRNA expression of BDNF was significantly restored in the Scop + Suff/H groups (Fig. 5D, F (3,13) = 5.398, Scop + Veh vs. Scop + Suff/H). The TrkB receptor shows a strong affinity to the mature form of BDNF 45 . To examine the effect of suffrutiocosol A on mBDNF restoration, we conducted western blotting to mBDNF expression. The protein level of mBDNF was decreased in Scop + Veh groups while significantly recovered in Scop + Suff/H groups (Fig. 5E, F (3,14) = 6.504, p < 0.05). These findings indicate that suffruticosol A ameliorates mBDNF levels against scopolamine treatment.
Suffruticosol A protects the scopolamine-induced impairment of long-term potentiation in the hippocampus. Long-term potentiation (LTP), a long-lasting enhancement of synaptic transmission, is supposed to be the type of synaptic plasticity that motivates hippocampal learning and memory functions 47 . We investigated whether the bath application of suffruticosol A could restore the LTP impairment induced by the scopolamine in the hippocampus. We applied a single dose of suffruticosol A (40 μM) to the slices of ( www.nature.com/scientificreports/ hippocampal tissues, which were perfused with artificial cerebrospinal fluid (aCSF) containing either DMSO vehicle or scopolamine (100 μM). In the control group, the field excitatory postsynaptic potential (fEPSP) was significantly increased and maintained for 60 min after TBS stimulation (Fig. 6). In the scopolamine-treated group (Scop + Veh), LTP was completely impaired (Fig. 6C, F (2,16) = 8.229, p < 0.001). Remarkably, the combined treatment of suffruticosol A restored the scopolamine-induced LTP impairment (Fig. 6C, p < 0.05). These findings suggested that suffruticosol A protects against the scopolamine-induced impairment of LTP in the hippocampus. This effect could be attributed to suffruticosol A acting as a synaptic mechanism that contributes to the neuroprotective properties of suffruticosol A in hippocampal memory.

Discussion
Paeonia lactiflora is a medicinal herb commonly used in traditional Chinese medicine. This herb has been known to prevent blood clotting, carcinogenesis, and inflammation partly due to its strong antioxidant capacity 48,49 .
In contrast, the seeds of this herd gained less attention for medicinal purposes. In this study, we found that the seeds of P. lactiflora have a complex structure of resveratrol, called suffruticosol A, and this chemical showed ameliorative effects against scopolamine-induced dementia. Resveratrol is an intriguing chemical because it  www.nature.com/scientificreports/ has a polyphenolic antioxidant and can be easily found in many plants, such as grapes, berries, and nuts 50 . Since its structure was identified in 1940 from the roots of white hellebore, it has gained eminence associated with the 'French Paradox' related to the consumption of red wine. Consuming red wines refers to the very low incidence of and mortality rates from cardiovascular diseases in the French 51 . Recently, resveratrol derivatives from grapes, Vitis vinifera, also have shown strong neuronal amelioration activities in dementia 52 . In this study, we found an interesting derivative structure of resveratrol isolated from the seeds of P. lactiflora. Suffruticosol A abundant in the seeds of P. lactiflora may be used as a therapeutic agent against dementia partially due to its neuronal amelioration effects. In AD patients, cholinergic impairments usually occur in the hippocampus, nucleus basalis of Meynert, and cortex 53 , which contributes to memory deficits 54 . An animal model of scopolamine-induced memory deficits, similar to the AD 9 . Scopolamine is a non-selective antagonist of a muscarinic receptor (M receptor) that blocks a neurotransmitter, acetylcholine (ACh) 55 . The M1 receptors are predominant in the cortex and hippocampus 56 . Blocking of M1 receptors causes damage to the hippocampus through the excessive release of ACh 57 , and reduces the long-term potentiation (LTP) 58 . The impaired hippocampal LTP is related to cognitive deficit because of its role in the learning and memory process [58][59][60] . Thus, scopolamine treatment induces a decrease in the cholinergic system and a loss of memory which resembles the AD patients 9,61 . Furthermore, scopolamine treatments do not necessitate complex surgical procedures, making this chemical a common choice for investigating therapeutic agents for neurodegenerative diseases.
The scopolamine-induced memory deficit model exhibits similarities in the downregulated brain-derived neurotrophic factor (BDNF) levels in AD patients 62 . BDNF signaling has a crucial role in AD, as evidenced by the low mRNA and protein expression levels of BDNF observed in AD patients 63,64 . Post-mortem examination of brains from AD patients revealed a loss of BDNF expression in both reactive microglia and neurons, which contained massive neurofibrillary tangles compared to normal neurons 65 . Prolonged depletion of ACh in the nucleus basalis downregulates ACh muscarinic receptor activation 44 . This downregulation depletes BDNF Values are expressed as means ± SEM (n = 6 for control and scopolamine, n = 7 for suffruticosol A with scopolamine; * p < 0.05, ** p < 0.01; one-way ANOVA with Tukey's multiple comparisons test). www.nature.com/scientificreports/ mRNA transcription and mature BDNF (mBDNF) protein level 44 . Scopolamine treatment hinders non-selective muscarinic receptor activation which induces a reduction of BDNF levels. Furthermore, scopolamine reduces the expression level of the mBDNF receptor, TrkB 66 . Restoring cholinergic function could re-establish mBDNF level. The mRNA level of related signals such as Akt and Creb1 may indicate the increase of mBDNF is derived from the cAMP response elements-binding protein (CREB) signaling cascade, with limited evidence. Based on our study, the administration of suffruticosol A postponed the deficit of cholinergic function and impairment of cognition and memory. The administration demonstrated improvements in long-term memory, partially attributed to the restoration of cholinergic function and BDNF signaling. The cholinergic functions induce neurogenesis in the hippocampus via the BDNF signaling pathway, which is necessary for long-term potentiation (LTP)-memory formation. LTP refers to a long-lasting increase in the effectiveness of excitatory synaptic transmission. Hippocampal LTP is commonly responsible for a cellular signaling cascade of learning and memory 59,60 . BDNF and glutamate are mostly working on memory functions 67 . BDNF is tightly related to the LTP by directly working on depolarizing neurons by increasing glutamatergic transmission for inducing phosphorylation of NMDA signaling via its TrkB receptors 68 . The BDNF positively controls LTP, promoting memory formation at the cellular and molecular levels. In addition, treatments of recombinant BDNF rescued impaired LTP in BDNF-mutant mice 69 . The LTP formation, which is connected to presynaptic neurons, depends on BDNF protein synthesis 69,70 . Thus, the BDNF regulates the translation of protein synthesis through several intracellular signaling pathways, related to cell growth, survival, differentiation, and intracellular trafficking via Akt and PI3K signaling. Our study presented that the central administration of suffruticosol A into the brain restores the deficits, impaired by scopolamine treatments, in hippocampal LTP partly through inducing BDNF activation. These recovered activities and the enhanced capability of LTP in the brain might contribute to neuroprotection in neurodegenerative diseases.

Conclusion
The treatments of suffruticosol A enhanced neural activity in cell lines, restored LTP-memory formation, recovered the cholinergic system, and increased memory and cognitive behaviors through partly BDNF signaling. These findings suggest that suffruticosol A might be considered a therapy for neurodegenerative disease.